Adjuvant formulation comprising a submicron oil droplet emulson

ABSTRACT

An adjuvant composition, comprising a metabolizable oil and an emulsifying agent, wherein the oil and the detergent are present in the form of an oil-in-water emulsion having oil droplets substantially all of which are less than 1 micron in diameter. In preferred embodiments, the emulsifying agent is also an immunostimulating agent, such as a lipophilic muramyl peptide. Alternatively, an immunostimulating agent separate from the emulsifying agent can be used.

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 357,035, filed May 25, 1989, which is herein incorporated byreference.

INTRODUCTION TECHNICAL FIELD

[0002] This invention relates generally to immunological adjuvants foruse in increasing efficiency of vaccines and is particularly directed toadjuvants comprising oil-in-water emulsions.

BACKGROUND

[0003] The emergence of new subunit vaccines created by recombinant DNAtechnology has intensified the need for safe and effective adjuvants.Traditional live anti-viral vaccines require no adjuvants. Killed virusvaccines are generally much more immunogenic than subunit vaccines andcan be effective with no adjuvant or with adjuvants that have limitedability to stimulate immune responses. The new, recombinant DNA-derivedsubunit vaccines, while offering significant advantages over thetraditional vaccines in terms of safety and cost of production,generally represent isolated proteins or mixtures of proteins that havelimited immunogenicity compared to whole viruses. Such materials arereferred to generally in this specification as molecular antigens, todistinguish them from the whole organisms (and parts thereof) that werepreviously used in vaccines. These vaccines will require adjuvants withsignificant immunostimulatory capabilities to reach their full potentialin preventing disease.

[0004] Currently, the only adjuvants approved for human use in theUnited States are aluminum salts (alum). These adjuvants have beenuseful for some vaccines including hepatitis B, diphtheria, polio,rabies and influenza, but may not be useful for others, especially ifstimulation of cell-mediated immunity is required for protection.Reports indicate that alum failed to improve the effectiveness ofwhooping cough and typhoid vaccines and provided only a slight effectwith adenovirus vaccines. Problems with aluminum salts include inductionof granulomas at the injection site and lot-to-lot variation of alumpreparations.

[0005] Complete Freund's adjuvant (CFA) is a powerful immunostimulatoryagent that has been used successfully with many antigens on anexperimental basis. CFA is comprised of three components: a mineral oil,an emulsifying agent such as Arlacel A, and killed mycobacteria such asMycobacterium tuberculosis. Aqueous antigen solutions are mixed withthese components to create a water-in-oil emulsion. CFA causes severeside effects, however, including pain, abscess formation, and fever,which prevent its use in either human or veterinary vaccines. The sideeffects are primarily due to the host's reactions to the mycobacterialcomponent of CFA. Incomplete Freund's adjuvant (IFA) is similar to CFAwithout the bacterial component. While not approved for use in theUnited States, IFA has been useful for several types of vaccines inother countries. IFA has been used successfully in humans with influenzaand polio vaccines and with several animal vaccines including rabies,canine distemper, and foot-and-mouth disease. Experiments have shownthat both the oil and emulsifier used in IFA can cause tumors in mice,indicating that an alternative adjuvant would be a better choice forhuman use.

[0006] Muramyl dipeptide (MDP) represents the minimal unit of themycobacterial cell wall complex that generates the adjuvant activityobserved with CFA; see Ellouz et al. (1974) Biochem. Biophys. Res.Comm., 59:1317. Many synthetic analogues of MDP have been generated thatexhibit a wide range of adjuvant potency and side effects (reviewed inChedid et al. (1978) Prog. Allergy, 25:63). Three analogues that may beespecially useful as vaccine adjuvants are threonyl derivatives of MDP,see Byars et al. (1987) Vaccine, 5:223; n-butyl derivatives of MDP, seeChedid et al. (1982) Infect. and Immun., 35:417; and lipophilicderivative of muramyl tripeptide, see Gisler et al. (1981) inImmunomodulations of Microbial Products and Related Synthetic Compounds,Y. Yamamura and S. Kotani, eds., Excerpta Medica, Amsterdam, p. 167.These compounds effectively stimulate humoral and cell-mediated immunityand exhibit low levels of toxicity.

[0007] One promising lipophilic derivative of MDP isN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1,2-dipalmitoyl-sn-glycero-3-3(hydroxyphosphoryl-oxy)]ethylamide(MTP-PE). This muramyl tripeptide has phospholipid tails that allowassociation of the hydrophobic portion of the molecule with a lipidenvironment while the muramyl peptide portion associates with theaqueous environment. Thus the MTP-PE itself can act as an emulsifyingagent to generate stable oil in water emulsions.

[0008] Original mouse experiments in the laboratories of the presentinventors with MTP-PE showed that this adjuvant was effective instimulating anti-HSV gD antibody titers against herpes simplex virus gDantigen and that effectiveness was vastly improved if the MTP-PE and gDwere delivered in oil (IFA) rather than in aqueous solution. Since IFAis not approved for human use, other oil delivery systems wereinvestigated for MTP-PE and antigen. An emulsion of 4% squalene with0.008% Tween 80 and HSV gD gave very good immunity in the guinea pig.This formulation, MTP-PE-LO (low oil), was emulsified by passing througha hypodermic needle and was quite unstable. Nevertheless, thisformulation gave high antibody titers in the guinea pig and goodprotection in a HSV challenge of immunized guinea pigs. The formulationwas most effective when delivered in the footpad but also gavereasonable antibody titers and protection when deliveredintramuscularly. These data have appeared in 2 publications(Sanchez-Pescador et al., J. Immunology 141, 1720-1727, 1988 andTechnological Advances in Vaccine Development, Lasky et al., ed., AlanR. Liss, Inc., p. 445-469, 1988). The MTP-PE-LO formulation was alsoeffective in stimulating the immune response to the yeast-produced HIVenvelope protein in guinea pigs. Both ELISA antibody titers and virusneutralizing antibody titers were stimulated to a high level with theMTP-PE formulation. However, when the same formulation was tested inlarge animals, such as goats and baboons, the compositions were not aseffective. The desirability of additional adjuvant formulations for usewith molecular antigens in humans and other large animals is evident.

SUMMARY OF THE INVENTION

[0009] Accordingly, it is an object of the present invention to providean adjuvant formulation suitable for stimulating immune responses tomolecular antigens in large mammals.

[0010] Surprisingly, it has been found that a satisfactory adjuvantformulation is provided by a composition comprising a metabolizable oiland an emulsifying agent, wherein the oil and the emulsifying agent arepresent in the form of an oil-in-water emulsion having oil dropletssubstantially all of which are less than 1 micron in diameter andwherein the composition exists in the absence of anypolyoxy-proplyene-polyoxyethylene block copolymer. Such block copolymerswere previously thought to be essential for the preparation of submicronoil-in-water emulsions. The composition can also contain animmunostimulating agent (which can be the same as the emulsifying agent,if an amphipathic immunostimulating agent is selected).

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0011] The present invention provides an adjuvant composition comprisinga metabolizable oil and an emulsifying agent, wherein the oil and theemulsifying agent are present in the form of an oil-in-water emulsionhaving oil droplets substantially all of which are less than 1 micron indiameter. Investigations in the laboratories of the present inventors,reported in detail in the examples that follow, show a surprisingsuperiority over adjuvant compositions containing oil and emulsifyingagents in which the oil droplets are significantly larger than thoseprovided by the present invention.

[0012] The individual components of the adjuvant compositions of thepresent invention are known, although such compositions have not beencombined in the same manner and provided in a droplet size of such smalldiameter. Accordingly, the individual components, although describedbelow both generally and in some detail for preferred embodiments, arewell known in the art, and the terms used herein, such as metabolizableoil, emulsifying agent, immunostimulating agent, muramyl peptide, andlipophilic muramyl peptide, are sufficiently well known to describethese compounds to one skilled in the art without further description.

[0013] One component of these formulations is a metabolizable, non-toxicoil, preferably one of 6 to 30 carbon atoms including, but not limitedto, alkanes, alkenes, alkynes, and their corresponding acids andalcohols, the ethers and esters thereof, and mixtures thereof. The oilmay be any vegetable oil, fish oil, animal oil or synthetically preparedoil which can be metabolized by the body of the subject to which theadjuvant will be administered and which is not toxic to the subject. Thesubject is an animal, typically a mammal, and preferably a human.Mineral oil and similar toxic petroleum distillate oils are expresslyexcluded from this invention.

[0014] The oil component of this invention may be any long chain alkane,alkene or alkyne, or an acid or alcohol derivative thereof either as thefree acid, its salt or an ester such as a mono-, or di- or triester,such as the triglycerides and esters of 1,2-propanediol or similarpoly-hydroxy alcohols. Alcohols may be acylated employing a mono- orpoly-functional acid, for example acetic acid, propanoic acid, citricacid or the like. Ethers derived from long chain alcohols which are oilsand meet the other criteria set forth herein may also be used.

[0015] The individual alkane, alkene or alkyne moiety and its acid oralcohol derivatives will have 6-30 carbon atoms. The moiety may have astraight or branched chain structure. It may be fully saturated or haveone or more double or triple bonds. Where mono or poly ester- orether-based oils are employed, the limitation of 6-30 carbons applies tothe individual fatty acid or fatty alcohol moieties, not the totalcarbon count.

[0016] Any metabolizable oil, particularly from an animal, fish orvegetable source, may be used herein. It is essential that the oil bemetabolized by the host to which it is administered, otherwise the oilcomponent may cause abscesses, granulomas or even carcinomas, or (whenused in veterinary practice) may make the meat of vaccinated birds andanimals unacceptable for human consumption due to the deleterious effectthe unmetabolized oil may have on the consumer.

[0017] Sources for vegetable oils include nuts, seeds and grains. Peanutoil, soybean oil, coconut oil, and olive oil, the most commonlyavailable, exemplify the nut oils. Seed oils include safflower oil,cottonseed oil, sunflower seed oil, sesame seed oil and the like. In thegrain group, corn oil is the most readily available, but the oil ofother cereal grains such as wheat, oats, rye, rice, teff, triticale andthe like may also be used.

[0018] The technology for obtaining vegetable oils is well developed andwell known. The compositions of these and other similar oils may befound in, for example, the Merck Index, and source materials on foods,nutrition and food technology.

[0019] The 6-10 carbon fatty acid esters of glycerol and1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. These products arecommercially available under the name NEOBEE® from PVO International,Inc., Chemical Specialties Division, 416 Division Street, Boongon, N.J.and others.

[0020] Oils from any animal source, may be employed in the adjuvants andvaccines of this invention. Animal oils and fats are usually solids atphysiological temperatures due to the fact that they exist astriglycerides and have a higher degree of saturation than oils from fishor vegetables. However, fatty acids are obtainable from animal fats bypartial or complete triglyceride saponification which provides the freefatty acids. Fats and oils from mammalian milk are metabolizable and maytherefore be used in the practice of this invention. The procedures forseparation, purification, saponification and other means necessary forobtaining pure oils from animal sources are well known in the art.

[0021] Most fish contain metabolizable oils which may be readilyrecovered. For example, cod liver oil, shark liver oils, and whale oilsuch as spermaceti exemplify several of the fish oils which may be usedherein. A number of branched chain oils are synthesized biochemically in5-carbon isoprene units and are generally referred to as terpenoids.Shark liver oil contains a branched, unsaturated terpenoids known assqualene, 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaenewhich is particularly preferred herein. Squalane, the saturated analogto squalene, is also a particularly preferred oil. Fish oils, includingsqualene and squalane, are readily available from commercial sources ormay be obtained by methods known in the art.

[0022] The oil component of these adjuvants and vaccine formulationswill be present in an amount from 0.5% to 20% by volume but preferablyno more than 15%, especially in an amount of 1% to 12%. It is mostpreferred to use from 1% to 4% oil.

[0023] The aqueous portion of these adjuvant compositions is bufferedsaline or, in preferred embodiments, unadulterated water. Because thesecompositions are intended for parenteral administration, it ispreferable to make up final buffered solutions used as vaccines so thatthe tonicity, i.e., osmolality, is essentially the same as normalphysiological fluids in order to prevent post-administration swelling orrapid absorption of the composition because of differential ionconcentrations between the composition and physiological fluids. It isalso preferable to buffer the saline in order to maintain a pHcompatible with normal physiological conditions. Also, in certaininstances, it may be necessary to maintain the pH at a particular levelin order to insure the stability of certain composition components suchas the glycopeptides.

[0024] Any physiologically acceptable buffer may be used herein, butphosphate buffers are preferred. Other acceptable buffers such asacetate, tris, bicarbonate, carbonate, or the like may be used assubstitutes for phosphate buffers. The pH of the aqueous component willpreferably be between 6.0-8.0.

[0025] However, when the adjuvant is initially prepared, unadulteratedwater is preferred as the aqueous component of the emulsion. Increasingthe salt concentration makes it more difficult to achieve the desiredsmall droplet size. When the final vaccine formulation is prepared fromthe adjuvant, the antigenic material can be added in a buffer at anappropriate osmolality to provide the desired vaccine composition.

[0026] The quantity of the aqueous component employed in thesecompositions will be that amount necessary to bring the value of thecomposition to unity. That is, a quantity of aqueous componentsufficient to make 100% will be mixed, with the other components listedabove in order to bring the compositions to volume.

[0027] A substantial number of emulsifying and suspending agents aregenerally used in the pharmaceutical sciences. These include naturallyderived materials such as gums from trees, vegetable protein,sugar-based polymers such as alginates and cellulose, and the like.Certain oxypolymers or polymers having a hydroxide or other hydrophilicsubstituent on the carbon backbone have surfactant activity, forexample, povidone, polyvinyl alcohol, and glycol ether-based mono- andpoly-functional compounds. Long chain fatty-acid-derived compounds forma third substantial group of emulsifying and suspending agents whichcould be used in this invention. Any of the foregoing surfactants areuseful so long as they are non-toxic.

[0028] Specific examples of suitable emulsifying agents (also referredto as surfactants or detergents) which can be used in accordance withthe present invention include the following:

[0029] 1. Water-soluble soaps, such as the sodium, potassium, ammoniumand alkanol-ammonium salts of higher fatty acids (C₁₀-C₂₂), and,particularly sodium and potassium tallow and coconut soaps.

[0030] 2. Anionic synthetic non-soap detergents, which can berepresented by the water-soluble salts of organic sulfuric acid reactionproducts having in their molecular structure an alkyl radical containingfrom about 8 to 22 carbon atoms and a radical selected from the groupconsisting of sulfonic acid and sulfuric acid ester radicals. Examplesof these are the sodium or potassium alkyl sulfates, derived from tallowor coconut oil; sodium or potassium alkyl benzene sulfonates; sodiumalkyl glyceryl ether sulfonates; sodium coconut oil fatty acidmonoglyceride sulfonates and sulfates; sodium or potassium salts ofsulfuric acid esters of the reaction product of one mole of a higherfatty alcohol and about 1 to 6 moles of ethylene oxide; sodium orpotassium alkyl phenol ethylene oxide ether sulfonates, with 1 to 10units of ethylene oxide per molecule and in which the alkyl radicalscontain from 8 to 12 carbon atoms; the reaction product of fatty acidsesterified with isethionic acid and neutralized with sodium hydroxide;sodium or potassium salts of fatty acid amide of a methyl tauride; andsodium and potassium salts of SO₃-sulfonated C₁₀-C₂₄ α-olefins.

[0031] 3. Nonionic synthetic detergents made by the condensation ofalkylene oxide groups with an organic hydrophobic compound. Typicalhydrophobic groups include condensation products of propylene oxide withpropylene glycol, alkyl phenols, condensation product of propylene oxideand ethylene diamine, aliphatic alcohols having 8 to 22 carbon atoms,and amides of fatty acids.

[0032] 4. Nonionic detergents, such as amine oxides, phosphine oxidesand sulfoxides, having semipolar characteristics. Specific examples oflong chain tertiary amine oxides include dimethyldodecylamine oxide andbis-(2-hydroxyethyl) dodecylamine. Specific examples of phosphine oxidesare found in U.S. Pat. No. 3,304,263 which issued Feb. 14, 1967, andinclude dimethyldodecyl-phosphine oxide and dimethyl-(2hydroxydodecyl)phosphine oxide.

[0033] 5. Long chain sulfoxides, including those corresponding to theformula R¹—SO—R² wherein R¹ and R² are substituted or unsubstitutedalkyl radicals, the former containing from about 10 to about 28 carbonatoms, whereas R² contains from 1 to 3 carbon atoms. Specific examplesof these sulfoxides include dodecyl methyl sulfoxide and 3-hydroxytridecyl methyl sulfoxide.

[0034] 6. Ampholytic synthetic detergents, such as sodium3-dodecylaminopropionate and sodium 3-dodecylaminopropane sulfonate.

[0035] 7. Zwitterionic synthetic detergents, such as3-(N,N-dimethyl-N-hexadecylammonio) propane-1-sulfonate and3-(N,N-dimethyl-N-hexadecylammonio)-2-hydroxy propane-1-sulfonate.

[0036] Additionally, all of the following types of emulsifying agentscan be used in a composition of the present invention: (a) soaps (i.e.,alkali salts) of fatty acids, rosin acids, and tall oil; (b) alkyl arenesulfonates; (c) alkyl sulfates, including surfactants with bothbranched-chain and straight-chain hydrophobic groups, as well as primaryand secondary sulfate groups; (d) sulfates and sulfonates containing anintermediate linkage between the hydrophobic and hydrophilic groups,such as the fatty acylated methyl taurides and the sulfated fattymonoglycerides; (e) long-chain acid esters of polyethylene glycol,especially the tall oil esters; (f) polyethylene glycol ethers ofalkylphenols; (g) polyethylene glycol ethers of long-chain alcohols andmercaptans; and (h) fatty acyl diethanol amides. Since surfactants canbe classified in more than one manner, a number of classes ofsurfactants set forth in this paragraph overlap with previouslydescribed surfactant classes.

[0037] There are a number of emulsifying agents specifically designedfor and commonly used in biological situations. For example, a number ofbiological detergents (surfactants) are listed as such by Sigma ChemicalCompany on pages 310-316 of its 1987 Catalog of Biochemical and OrganicCompounds. Such surfactants are divided into four basic types: anionic,cationic, zwitterionic, and nonionic. Examples of anionic detergentsinclude alginic acid, caprylic acid, cholic acid, 1-decanesulfonic acid,deoxycholic acid, 1-dodecanesulfonic acid, N-lauroylsarcosine, andtaurocholic acid. Cationic detergents include dodecyltrimethylammoniumbromide, benzalkonium chloride, benzyldimethylhexadecyl ammoniumchloride, cetylpyridinium chloride, methylbenzethonium chloride, and4-picoline dodecyl sulfate. Examples of zwitterionic detergents include3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (commonlyabbreviated CHAPS),3-[(cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate(generally abbreviated CHAPSO),N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, andlyso-α-phosphatidylcholine. Examples of nonionic detergents includedecanoyl-N-methylglucamide, diethylene glycol monopentyl ether,n-dodecyl β-D-glucopyranoside, ethylene oxide condensates of fattyalcohols (e.g., sold under the trade name Lubrol), polyoxyethyleneethers of fatty acids (particularly C₁₂-C₂₀ fatty acids),polyoxyethylene sorbitan fatty acid ethers (e.g., sold under the tradename Tween), and sorbitan fatty acid ethers (e.g., sold under the tradename Span).

[0038] A particularly useful group of surfactants are the sorbitan-basednon-ionic surfactants. These surfactants are prepared by dehydration ofsorbitol to give 1,4-sorbitan which is then reacted with one or moreequivalents of a fatty acid. The fatty-acid—substituted moiety may befurther reacted with ethylene oxide to give a second group ofsurfactants.

[0039] The fatty-acid-substituted sorbitan surfactants are made byreacting 1,4-sorbitan with a fatty acid such as lauric acid, palmiticacid, stearic acid, oleic acid, or a similar long chain fatty acid togive the 1,4-sorbitan mono-ester, 1,g-sorbitan sesquiester or1,4-sorbitan triester. The common names for these surfactants include,for example, sorbitan monolaurate, sorbitan monopalmitate, sorbitanmonoestearate, sorbitan monooleate, sorbitan sesquioleate, and sorbitantrioleate. These surfactants are commercially available under the nameSPAN® or ARLACEL®, usually with a letter or number designation whichdistinguishes between the various mono, di- and triester substitutedsorbitans.

[0040] SPAN® and ARLACEL® surfactants are hydrophilic and are generallysoluble or dispersible in oil. They are also soluble in most organicsolvents. In water they are generally insoluble but dispersible.Generally these surfactants will have a hydrophilic-lipophilic balance(HLB) number between 1.8 to 8.6. Such surfactants can be readily made bymeans known in the art or are commercially available from, for example,ICI America's Inc., Wilmington, Del. under the registered mark ATLAS®.

[0041] A related group of surfactants comprises polyoxyethylene sorbitanmonoesters and polyoxyethylene sorbitan triesters. These materials areprepared by addition of ethylene oxide to a 1,4-sorbitan monester ortriester. The addition of polyoxyethylene converts the lipophilicsorbitan mono- or triester surfactant to a hydrophilic surfactantgenerally soluble or dispersible in water and soluble to varying degreesin organic liquids.

[0042] These materials, commercially available under the mark TWEEN®,are useful for preparing oil-in-water emulsions and dispersions, or forthe solubilization of oils and making anhydrous ointments water-solubleor washable. The TWEEN® surfactants may be combined with a relatedsorbitan monester or triester surfactants to promote emulsion stability.TWEEN® surfactants generally have a HLB value falling between 9.6 to16.7. TWEEN® surfactants are commercially available from a number ofmanufacturers, for example ICI America's Inc., Wilmington, Del. underthe registered mark ATLAS® surfactants.

[0043] A third group of non-ionic surfactants which could be used aloneor in conjunction with SPAN®, ARLACEL® and TWEEN® surfactants are thepolyoxyethylene fatty acids made by the reaction of ethylene oxide witha long-chain fatty acid. The most commonly available surfactant of thistype is solid under the name MYRJ® and is a polyoxyethylene derivativeof stearic acid. MYRJ® surfactants are hydrophilic and soluble ordispersible in water like TWEEN® surfactants. The MYRJ® surfactants maybe blended with TWEEN® surfactants or with TWEEN®/SPAN® or ARLACEL®surfactant mixtures for use in forming emulsions. MYRJ® surfactants canbe made by methods known in the art or are available commercially fromICI America's Inc.

[0044] A fourth group of polyoxyethylene based non-ionic surfactants arethe polyoxyethylene fatty acid ethers derived from lauryl, acetyl,stearyl and oleyl alcohols. These materials are prepared as above byaddition of ethylene oxide to a fatty alcohol. The commercial name forthese surfactants is BRIJ®. BRIJ® surfactants may be hydrophilic™orlipophilic depending on the size of the polyoxyethylene moiety in thesurfactant. While the preparation of these compounds is available fromthe art, they are also readily available from such commercial sources asICI America's Inc.

[0045] Other non-ionic surfactants which could potentially be used inthe practice of this invention are for example: polyoxyethylene, polyolfatty acid esters, polyoxyethylene ether, polyoxypropylene fatty ethers,bee's wax derivatives containing polyoxyethylene, polyoxyethylenelanolin derivative, polyoxyethylen fatty glycerides, glycerol fatty acidesters or other polyoxyethylene acid alcohol or ether derivatives oflong-chain fatty acids of 12-22 carbon atoms.

[0046] As the adjuvant and the vaccine formulations of this inventionare intended to be multi-phase systems, it is preferable to choose anemulsion-forming non-ionic surfactant which has an HLB value in therange of about 7 to 16. This value may be obtained through the use of asingle non-ionic surfactant such as a TWEEN® surfactant or may beachieved by the use of a blend of surfactants such as with a sorbitanmono, di- or triester based surfactant; a sorbitan ester polyoxyethylenefatty acid; a sorbitan ester in combination with a polyoxyethylenelanolin derived surfactant; a sorbitan ester surfactant in combinationwith a high HLB polyoxyethylene fatty ether surfactant, or apolyethylene fatty ether surfactant or polyoxyethylene sorbitan fattyacid.

[0047] It is more preferred to use a single non-ionic surfactant, mostparticularly a TWEEN® surfactant, as the emulsion stabilizing non-ionicsurfactant in the practice of this invention. The surfactant namedTWEEN® 80, otherwise known as polysorbate 80 for polyoxyethlyene 20sorbitan monooleate, is the most preferred of the foregoing surfactants.

[0048] Sufficient droplet size reduction can usually be effected byhaving the surfactant present in an amount of 0.02% to 2.5% by weight(w/w). An amount of 0.05% to 1% is preferred with 0.01 to 0.5% beingespecially preferred.

[0049] The manner in which the droplet size of the invention is reachedis not important to the practice of the present invention. One manner inwhich submicron oil droplets can be obtained is by use of a commercialemulsifiers, such as model number 11OY available from Microfluidics,Newton, Mass. Examples of other commercial emulsifiers include GaulinModel 30CD (Gaulin, Inc., Everett, Mass.) and Rainnie Minilab Type 8.30H(Miro Atomizer Food and Dairy, Inc., Hudson, Wis.). These emulsifiersoperate by the principle of high shear forces developed by forcingfluids through small apertures under high-pressure. When the model 11OYis operated at 5,000-30,000 psi, oil droplets having diameters of100-750 nm are provided.

[0050] The size of the oil droplets can be varied by changing the ratioof detergent to oil (increasing the ratio decreases droplet size),operating pressure (increasing operating pressure reduces droplet size),temperature (increasing temperature decreases droplet size), and addingan amphipathic immunostimulating agent (adding such agents decreasesdroplet size). Actual droplet size will vary with the particulardetergent, oil, and immunostimulating agent (if any) and with theparticular operating conditions selected. Droplet size can be verifiedby use of sizing instruments, such as the commercial Sub-Micron ParticleAnalyzer (Model N4MD) manufactured by the Coulter Corporation, and theparameters can be varied using the guidelines set forth above untilsubstantially all droplets are less than 1 micron in diameter,preferably less than 0.8 microns in diameter, and most preferably lessthan 0.5 microns in diameter. By substantially all is meant at least 80%(by number), preferably at least 90%, more preferably at least 95%, andmost preferably at least 98%. The particle size distribution istypically Gaussian, so that the average diameter is smaller than thestated limits.

[0051] The present invention is practiced by preparing an oil emulsionin the absence of other components previously taught in the prior art tobe used with submicron emulsions for satisfactory immunogenicity, namelypolyoxypropylene-polyoxyethlyne block polymers such as those describedfor use with adjuvants in U.S. Pat. No. 4,772,466 and 4,770,874 and inEuropean Patent Application 0 315 153 A2.

[0052] An adjuvant composition of the invention consists essentially ofa metabolizable oil in water and an emulsifying agent other than than aPOP-POE copolymer. The emulsifying agent need not have any specificimmunostimulating activity, since the oil composition by itself canfunction as an adjuvant when the oil droplets are in the submicronrange. However, increased immunostimulating activity can be provided byincluding any of the known immunostimulating agents in the composition.These immunostimulating agents can either be separate from theemulsifying agent and the oil or the immunostimulating agent and theemulsifying agent can be one and the same molecule. Examples of theformer situation include metabolizable oils mixed with killedmycobacteria, such as Mycobacterium tuberculosis, and subcellularcomponents thereof. Additional immunostimulating substances include themuramyl peptides that are components of the cell walls of such bacteria.A number of preferred muramyl peptides are listed below. Examples of thejoint emulsifying agent/immunostimulating agent are the lipophilicmuramyl peptides described in the two Sanchez-Pescador et al.publications cited above. These materials comprise the basicN-acetylmuramyl peptide (a hydrophilic moiety) that acts as animmunostimulating group, but also include a lipophilic moiety thatprovides surface-active characteristics to the resulting compound. Suchcompounds, as well as other types of amphipathic immunostimulatingsubstances, act as both immunostimulating agents and emulsifying agentsand are preferred in the practice of the present invention. In addition,it is also possible to practice the present invention by using aamphiphatic immunostimulating substance in combination with a secondimmunostimulating substance that is not amphipathic. An example would beuse of a lipophilic muramyl peptide in combination with an essentiallyunsubstituted (i.e., essentially hydrophilic) muramyl dipeptide.

[0053] The preferred immune-response-stimulating muramyl peptides (ormore accurately glycopeptides) of this invention are a group ofcompounds related to and generally derived fromN-acetylmuramyl-L-alanyl-D-isoglutamine, which was determined by Ellouzet al. (1974) Biochem. & Biophys. Res. Comm., 59(4): 1317, to be thesmallest effective unit possessing immunological adjuvant activity in M.tuberculosis, the mycobacterial component of Freund's complete adjuvant.A number of dipeptide- and polypeptide-substituted muramic acidderivatives were subsequently developed and found to haveimmunostimulating activity.

[0054] Though these glycopeptides are a diverse group of compounds, theycan be generally represented by Formula I below:

[0055] wherein the pyran ring oxygens are substituted by hydrogen,alkyl, or acyl or the like, or may be replaced by nitrogen-basedsubstituents, particularly the 6-position oxygen; the 2-amino group isan acyl group or some other amide; the lactyl side chain is modified,e.g., is ethyl or another two-position alkyl moiety; and the peptidefunction is a dipeptide or polypeptide, which may be furtherderivatized. Furanosyl analogues of the pyranosyl compounds also haveimmunopotentiating activity and are useful in this invention.

[0056] Among the glycopeptides of this invention are those disaccharidesand tetrasaccharides linked by meso-α,ε-diaminopimelic acid such asdescribed in U.S. Pat. Nos. 4,235,771 and 4,186,194.

[0057] Immune response stimulating glycopeptides which may be used inthe practice of this invention are disclosed in U.S. Pat. Nos.4,094,971; 4,101,536; 4,153,684; 4,235,771; 4,323,559; 4,327,085;4,185,089; 4,082,736; 4,369,178; 4,314,998 and 4,082,735; and 4,186,194.The glycopeptides disclosed in these patents are incorporated herein byreference and made a part hereof as if set out in full herein. Thecompounds of Japanese patent application Nos. JP 40792227, JP 4079228,and JP 41206696 would also be useful in the practice of this invention.

[0058] Methods for preparing these compounds are disclosed andwell-known in the art. Preparative process exemplification can be foundin U.S. Pat. Nos. 4,082,736 and 4,082,735. Additionally, similarpreparative processes may be found in the U.S. patents referenced in thepreceding paragraph.

[0059] Preferred glycopeptides are those having the Formula II

[0060] wherein

[0061] R is an unsubstituted or substituted alkyl radical containingfrom 1 to 22 carbon atoms, or an unsubstituted or substituted arylradical containing from 6 to 10 carbon atoms;

[0062] R¹and R² are the same or different and are hydrogen or an acylradical containing from 1 to 22 carbon atoms;

[0063] R³ is hydrogen, alkyl of 1 to 22 carbons, or aryl of 7 to 10carbon atoms;

[0064] R³ is hydrogen or alkyl;

[0065] n is 0 or 1;

[0066] X and Z are independently alanyl, valyl, leucyl, isoleucyl,α-aminobutyryl, threonyl, methionyl, cysteinyl, glutamyl, glutaminyl,isoglutamyl, isoglutaminyl, aspartyl, phenylalanyl, tyrosyl, lysyl,ornithinyl, arginyl, histidyl, asparaginyl, prolyl, hydroxyprolyl,seryl, or glycyl;

[0067] R⁵ is an optionally esterified or amidated carboxyl group of theterminal amino acid; and

[0068] Y is —NHCHR⁶CH₂CH₂CO—, wherein R⁶ is an optionally esterified oramidated carboxyl group.

[0069] Alkyl is a straight or branched radical comprised of 1 to 7carbon atoms unless otherwise specified, exemplified by methyl, ethyl,propyl, butyl, pentyl, hexyl or heptyl or an isomer. Lower alkyl is aradical of 1 to 4 carbon atoms.

[0070] An optionally esterified or amidated carboxyl group is thecarboxyl group itself or a carboxyl group esterified with a loweralkanol, such as methanol, ethanol, propanol, butanol, or the carbamoylgroup, which, on the nitrogen atom, is unsubstituted or monosubstitutedor di-substituted by alkyl, especially lower alkyl, aryl, particularlyphenyl, or arylalkyl, particularly benzyl. The carbamoyl group may alsobe substituted with an alkylidene radical such as butylidene orpentylidene radical. In addition, the carbamoyl group R⁵ may also besubstituted with a carbamoylmethyl group on the nitrogen atom.

[0071] Particularly preferred compounds are those of Formula II whereinR and R¹ are the same or different and are hydrogen or an acyl radicalcontaining from 1 to 22 carbon atoms; R² is methyl; R³ is hydrogen; X isL-alanyl, Y is D-isoglutaminyl, and n is 0.

[0072] A different preferred group of glycopeptides are the compounds ofFormula II wherein R and R¹ are hydrogen or acyl of 1 to 22 carbonatoms, R² is methyl, R² is hydrogen, R⁴ is methyl or butyl, and X isL-valyl, L-seryl, L-alanyl, L-threonyl or L-α--aminobutyryl.

[0073] Specific examples include the following compounds:

[0074] N-acetylmuramyl-L-α-aminobutyryl-D-isoglutamine;

[0075] 6-0-stearoyl-N-acetylmuramyl-L-α-aminobutyryl-D-isoglutamine;

[0076] N-acetylmuramyl-L-threonyl-D-isoglutamine;

[0077] N-acetylmuramyl-L-valyl-D-isoglutamine;

[0078] N-acetylmuramyl-L-alanyl-D-glutamine n-butyl ester;

[0079] N-acetyl-desmethyl-D-muramyl-L-alanyl-D-isoglutamine;

[0080] N-acetylmuramyl-L-alanyl-D-glutamine;

[0081] N-acetylmuramyl-L-seryl-D-isoglutamine;

[0082] N-acetyl(butylmuramyl)-L-α-aminobutyl-D-isoglutamine; and

[0083] N-acetyl(butylmuramyl)-L-alanyl-D-isoglutamine.

[0084] An effective amount of immunostimulating glycopeptide is thatamount which effects an increase in antibody titer level whenadministered in conjunction with an antigen over that titer levelobserved when the glycopeptide has not been co-administered (typicallyin the range of 0.0001 to 10% of the total composition). As can beappreciated, each glycopeptide may have an effective dose range that maydiffer from the other glycopeptides. Therefore, a single dose rangecannot be prescribed which will have a precise fit for each possibleglycopeptide within the scope of this invention. However, as a generalrule, the glycopeptide will preferably be present in the vaccine in anamount of between 0.001 and 5% (w/v). A more preferred amount is 0.01 to3% (w/v).

[0085] Most of the immunostimulating glycopeptides discussed above areessentially hydrophilic compounds. Accordingly, they are intended foruse with a separate emulsifying agent (which can be, as discussed above,also an immunostimulating agent). In some case, the above-describedcompounds have a lipophilic character, such as the compounds comprisingfatty acid substituents and/or aryl substituents on the sugar moiety,particularly those containing one or more acyl radicals containing from14 to 22 carbon atoms, particularly those containing more than 1 suchacyl substituent. However, it is also possible to achieve lipophiliccharacter in a muramyl peptide by providing a lipid moiety linkedthrough the carboxylate group or side chains of the peptide moiety. Inparticular, lipid groups joined to the peptide moiety through theterminal carboxylate group represent a preferred grouping of compounds.This linkage can readily be provided either directly, such as by formingan ester linkage between the terminal carboxylate and a fatty alcoholcontaining from 14 to 22 carbon atoms, or by using a bifunctionallinking group, such as ethanolamine, to link the carboxylate througheither a ester or amide linkage to a lipid. Particularly preferred inthis embodiment of the invention are phospholipids, as the phosphategroups provide a readily linkable functional group.Diacylphospho-glycerides provide one such readily linkablephospho-lipid. Phosphatidyl ethanolamine, a readily available, naturallyoccurring compound, can be easily linked to the terminal carboxylate ofthe peptide moiety through an amide bond. Other lipids to the terminalcarboxyl include acylglycerols, phosphatidyl choline, phosphatidylserine, phosphatidyl inositol, phosphatidylglycerol, cardiolipin, andsphingomyelin.

[0086] A number of preferred amphipathic immunostimulating peptides arethose having Formula III below:

[0087] wherein R, R¹-R⁴, X, Y, Z and n have the previously describedmeanings. L represents a lipid moiety, such as the lipid moietiesdescribed above.

[0088] In summary, the muramic acid moiety and the peptide moiety of themolecule together provide a hydrophilic moiety. A lipophilic moiety isalso present in the molecule, lipophilicity generally being provided bya long-chain hydrocarbon group, typically present in the form of a fattyacid. The fatty acid or other hydrocarbon-containing radical can beattached to a hydroxyl group of the sugar or can be linked to thepeptide portion of the molecule either directly, such as by reacting afatty acid with a free amino group present in the peptide moiety, orthrough a linking group, such as a hydroxyalkylamine that forms a linkbetween a carboxylic acid group of the peptide through amide bondformation and a functional group in a lipid, such as a phosphate group.Phospholipid moieties are particularly preferred for use in forminglipophilic muramyl peptides. A group of preferred compounds includemuramyl dipeptides and tripeptides linked to a phospholipid moietythrough a hydroxyalkylamine moiety. An example, and a particularlypreferred compound, isN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy)]ethylamide(abbreviated MTP-PE).

[0089] The adjuvant formulations are generally prepared from theingredients described above prior to combining the adjuvant with theantigen that will be used in the vaccine. The word antigen refers to anysubstance, including a protein or protein-poly-saccharide,protein-lipopolysaccharide, poly-saccharide, lipopolysaccharide, viralsubunit, whole virus or whole bacteria which, when foreign to the bloodstream of an animal, on gaining access to the tissue of such an animalstimulates the formation of specific antibodies and reacts specificallyin vivo or in vitro with a homologous antibody. Moreover, it stimulatesthe proliferation of T-lymphocytes with receptors for the antigen andcan react with the lymphocytes to initiate the series of responsesdesignated cell-mediated immunity.

[0090] A hapten is within the scope of this definition. A hapten is thatportion of an antigenic molecule or antigenic complex that determines itimmunological specificity. Commonly, a hapten is a peptide orpolysaccharide in naturally occurring antigens. In artificial antigensit may be a low molecular weight substance such as an arsanilic acidderivative. A hapten will react specifically in vivo or in vitro withhomologous antibodies or T-lymphocytes. Alternative descriptors areantigenic determinant, antigenic structural grouping and haptenicgrouping.

[0091] The formulation of a vaccine of the invention will employ aneffective amount of an antigen. That is, there will be included anamount of antigen which, in combination with the adjuvant, will causethe subject to produce a specific and sufficient immunological responseso as to impart protection to the subject from the subsequent exposureto virus, bacterium, fungus, mycoplasma, or parasite immunized against.

[0092] Antigens may be produced by methods known in the art or may bepurchased from commercial sources. For example, U.S. Pat. Nos.4,434,157, 4,406,885, 4,264,587, 4,117,112, 4,034,081, and 3,996,907,incorporated herein by reference, describe methods for preparingantigens for feline leukemia virus vaccines. Other antigens maysimilarly be prepared. Antigens within the scope of this inventioninclude whole inactivated virus particles, isolated virus proteins andprotein subunits, whole cells and bacteria, cell membrane and cell wallproteins, and the like. Vaccines of the invention may be used toimmunize birds and mammals against diseases and infection, includingwithout limitation cholera, diphtheria, tetanus, pertussis, influenza,measles, meningitis, mumps, plague, poliomyelitis, rabies, RockyMountain spotted fever, rubella, smallpox, typhoid, typhus, felineleukemia virus, and yellow fever.

[0093] No single dose designation can be assigned which will providespecific guidance for each and every antigen which may be employed inthis invention. The effective amount of antigen will be a function ofits inherent activity and purity. It is contemplated that the adjuvantcompositions of this invention may be used in conjunction with wholecell or virus vaccines as well as with purified antigens or proteinsubunit or peptide vaccines prepared by recombinant DNA techniques orsynthesis.

[0094] Since the adjuvant compositions of the invention are stable, theantigen and emulsion can mixed by simple shaking. Other techniques, suchas passing a mixture of the adjuvant and solution or suspension of theantigen rapidly through a small opening (such as a hypodermic needle)readily provides a useful vaccine composition.

[0095] The invention now being generally described, the same will bebetter understood by reference to the following detailed examples whichare provided by way of illustration and are not intended to be limitingof the invention unless so specified.

EXAMPLE 1 General Techniques

[0096] The following general techniques were used throughout theexamples that follow, except where noted:

[0097] Materials

[0098] MTP-PE was provided by CIBA-GEIGY (Basel, Switzerland). Squaleneand Tween 80 were obtained from Sigma Chemical Co. (St. Louis, Mo.). CFAand IFA were obtained from Gibco (Grand Island, N.Y.). Aluminumhydroxide (Rehsorptar) was obtained from Reheis Chemical Co. (BerkeleyHeights, N.J.).

[0099] Preparation of Emulsions

[0100] Method 1—Syringe and needle. A mixture consisting of 4% squalene,0.008% Tween 80, 250 μg/ml MTP-PE and antigen in phosphate bufferedsaline (PBS) was passed through a 23 gauge needle 6 times. This emulsionconsisted of oil droplet sizes in the range of 10 microns and is termedMTP-PE-LO.

[0101] Method 2—Kirkland Emulsifier. The above mixture was passedthrough a Kirkland emulsifier five times. This emulsion consists of oildroplets primarily of 1-2 microns and is termed MTP-PE-LO-KE. TheKirkland emulsifier (Kirkland Products, Walnut Creek, Calif.) is asmall-scale version of the commercial knife-edged homogenizer (e.g.,Gaulin Model 30CD and Rainnie Minilab Type 8.30H) generating about 1000psi in the working chamber.

[0102] Method 3—Microfluidizer. Mixtures containing 0.3-18% squalene and0.2-1.0 mg/ml MTP-PE with or without Tween 80 were passed through theMicrofluidizer (Model No. 11OY, Microfluidics Newton, Mass.) at5,000-30,000 PSI. Typically, 50 ml of emulsion was mixed for 5 minutesor 100 ml for 10 minutes in the microfluidizer. The resulting emulsionsconsisted of oil droplets of 100-750 nm depending on squalene, MTP-PE,and detergent concentration and microfluidizer operating pressure andtemperature. This formulation is termed MTP-PE-LO-MF.

[0103] Antigen was added to the adjuvant formulations above afterpreparation. The antigen and emulsion were mixed by shaking. When usingCFA and IFA, antigen in PBS was mixed with an equal volume of either CFAor IFA The mixture was emulsified by passing through a hypodermic needleuntil a thick, emulsion was achieved.

[0104] Antigens

[0105] Herpes simplex virus (HSV) rgD2 is a recombinant protein producedgenetically engineered Chinese hamster ovary cells. This protein has thenormal anchor region truncated, resulting in a glycosylated proteinsecreted into tissue culture medium. The gD2 was purified in the CHOmedium to greater than 90% purity. Human immunodeficiency virus (HIV)env-2-3 is a recombinant form of the HIV enveloped protein produced ingenetically engineered Saccharomyces cerevisae. This protein representsthe entire protein region of HIV gp120 but is non-glycosylated anddenatured as purified from the yeast. HIV gp120 is a fully glycosylated,secreted form of gp120 produced in CHO cells in a fashion similar to thegD2 above.

[0106] Immunization of Animals

[0107] Mice were injected with the various adjuvant/antigen formulationsby intraperitoneal, intramuscular, or subcutaneous routes. Guinea pigswere immunized by footpad or intramuscular routes. Rabbits, goats, andbaboons were immunized by the intramuscular routes.

[0108] Analysis of Immune Response

[0109] Antibody titers against the immunizing antigen were determined byenzyme linked immunosorbent assay (ELISA).

EXAMPLE 2 MTP-PE-LO Formulation in Large Animals Comparative Example

[0110] A number of experiments were carried out, first with the HIV env2-3 antigen and later with the HSV gD protein, using the MTP-PE-LOformulation to stimulate immunity in large animals. These experimentsare outlined below.

[0111] 1. HIV env 2-3

[0112] a. Guinea pigs. Guinea pigs were immunized monthly with 50pg/dose of env 2-3 by either the footpad or intramuscular route. Thevaccine was administered with either the MTP-PE-LO formulation (4%Squalene, 0/008% Tween 80, 50 pg/dose MTP-PE) or absorbed to alum (0.7%aluminum hydroxide). Sera were collected one week after eachimmunization and analyzed for anti-env 2-3 antibody by ELISA. Theresults are shown in Table 1. The MTP-PE-LO formulation gave highanti-env 2-3 titers when delivered both intramuscularly and in thefootpad. In contrast, alum gave much lower antibody titers by bothroutes. This experiment illustrates the effectiveness of the MTP-PE-LOformulation in guinea pigs. TABLE 1 Comparison of Different Adjuvants,As a Function of Injection Route, In Eliciting Env 2-3 SpecificAntibodies^(a) Env 2-3 ELISA Titers djuvant Animal Immunization NumberGroup # Route Zero Two Three MTP-PE 839 FP  <<100^(c) 135,500 382,100 4%840 FP <<100 331,700 588,700 Squalene 0.008% 841 FP <<100 247,800330,900 Tween 842 FP <<100 108,100 570,300 843 FP <<100 65,00 — 844 FP<<100 25,000 — (average) (FP) (<<100) (152,000)        (468,000) MTP-PE845 IM <<100 12,300  19,600 4% 846 IM <<100 10,400  20,500 Squalene0.008% 847 IM <<100 29,700  80,000 Tween 848 IM <<100 447,000 640,000849 IM   350 10,600  78,700 850 IM <<100 340,000 — (average) (IM)(<<100) (142,000)        (168,000) Alum 863 FP <<100 <<100 nt 864 FP<<100 2,500  4,100 865 FP <<100 2,400  26,400 866 FP <<100 15,100103,900 867 FP <<100 2,200  8,800 868 FP <<100 6,500  44,500 (average)(FP) (<<100) (5,700)      (38,000) Alum 869 IM <<100 <<100 300 870 IM<<100 <<100 130 871 IM <<100 <<100  1,200 872 IM <<100 <<100 300 873 IM<<100 <<100 990 874 IM <<100 <<100 940 (average) (IM) (<<100)(<<100)      (640) Env 2-3 ELISA Titers djuvant Immunization NumberGroup Four Five Six Seven MTP-PE 343,100 401,800 338,000 382,700 4%542,300 392,900 359,000 292,100 Squalene 0.008% 301,100 285,800 334,400383,700 Tween 694,300 344,400 289,800 220,300 — — — — — — — — (470,000)(356,000) (330,000) (295,000) MTP-PE 23,800 15,100 20,000 27,300 4%43,600 44,800 121,100 42,000 Squalene 0.008% 136,800 156,000 144,500164,400 Tween 400,000 71,000 674,000 533,000 311,000 533,000 nt 200,000— — — — (183,000) (164,000) (240,000) (193,000) Alum nt nt nt nt 86,00047,700 21,000 16,000 80,400 83,500 39,200 4,500 124,100 107,100 56,70016,800 14,500 11,900 11,400 12,300 34,000 18,800 12,800 (68,000)(54,000) (28,000) (12,000) Alum 2,600 2,000 1,600 2,300 220 330 270 3004,300 4,900 3,000 1,600 900 920 770 1,700 41,100 79,800 27,900 15,50017,300 13,200 10,600 8,600 (11,000) (17,000) (7,000) (5,000)

[0113] b. Goats. Pairs of goats received 1 mg of env 2-3 on primaryimmunizations and 500 μg on secondary immunization with the MTP-PE-LOformulation containing various amounts of MTP-PE from 0 to 500 μg.Positive control animals received the primary immunization with CFA andthe secondary immunization with IFA. One group also received 100 μg env2-3 in the primary immunization followed by 50 μg in the secondaryimmunization with the MTP-PE-LO formulation containing 100 μg MTP-PE. Asshown in Table 2, both goats receiving Freund's adjuvant showed highantibody titers ranging from 2700 to 62,800. In contrast, most of thegoats receiving the MTP-PE-LO formulation were negative for anti-env 2-3antibody. Animals that did respond only developed titers in the 100-600range. These results are in stark contrast to the guinea pig data above.TABLE 2 Antibody Responses of Goats Immunized With Env 2-3 and VariousDoses of MTP-PE Adjuvant Env 2-3 ELISA Titer Formu- Animal Immunizationlation Number None One Two Freund's 2295 ^(b)<<100 43,200 62,800 2296<<100 2,700 7,500 ^(a)ST + 0 μg 2297 <<100 ^(c)<100 <100 MTP-PE 2298<<100 100 300 ST + 20 μg 2290 <<100 <100 <100 MTP-PE 2302 <<100 100 200ST + 50 μg 2301 <<100 <<100 <100 MTP-PE 2302 <<100 <<100 <100 ST + 100μg 2303 <<100 <<100 100 MTP-PE 2304 <<100 <<100 <100 ST + 250 μg 2305<<100 <100 600 MTP-PE 2306 <<100 <<100 <100 ST + 500 μg 2307 <<100 <100<100 MTP-PE 2308 <<100 <<100 <<100 ST + 100 μg 2309 200 500 200 MTP-PE2310 <<100 <100 <<100

[0114] c. Dogs. Beagle dogs were immunized with either 250 μg of env 2-3in MTP-PE-LO (100 μg MTP-PE) or with the MTP-PE-LO formulation alone atthree week intervals. Ten days after each immunization the animals werebled and anti-env 2-3 antibody titers were determined by ELISA. Table 3shows that the two dogs receiving env 2-3 plus adjuvant did developanti-env 23 titers, but these titers failed to reach the levels seen inguinea pigs (maximum titers 1700 and 6300 for the two immunizedanimals). In addition, these animals failed to develop virusneutralizing antibodies to either the homologous (SF2) or heterologous(BRU or Zr6) HIV strains. TABLE 3 ELISA and Neutralizing Antibody Titersof Sera From Beagle Dogs Immunized With Env 2-3 In MTP-PE-LOAdjuvant^(a) Env 2-3 Neutralization titers Animal Immunized ImmunizationELISA HIV- HIV- # with # titer HIV-SF2 BRU Zr6 1375 env 2-3 pre-^(b)<<100       ^(c)<20     <20 <20 bleed MTP-PE-LO 2 1,300 <20 <20 <20100 μg MTP-PE 3 1,700 <20 <20 <20 4 900 <20 <20 <20 5 400 <20 <20 <20 6300 <20 <20 <20 7 300 <20 <20 <20 1376 env 2-3 pre- <<100 <20 <20 <20bleed MTP-PE-LO 2 3,500 <20 <20 <20 100 μg MTP-PE 3 6,300 <20 <20 <20 45,100 <20 <20 <20 5 2,100 <20 <20 <20 6 2,200 <20 <20 <20 7 2,000 <20<20 <20 1377 MTP-PE-LO pre- <<100 <20 <20 <20 bleed O-MTP-PE 2 <<100 <20<20 <20 control 3 <<100 <20 <20 <20 5 <<100 <20 <20 <20 6 <<100 <20 <20<20 7 <<100 <20 <20 <20 1378 MTP-PE-LO pre- <<100 <20 <20 <20 bleedO-MTP-PE 2 <<100 <20 <20 <20 control 3 <<100 <20 <20 <20 4 <<100 <20 <20<20 5 <<100 <20 <20 <20 6 <<100 <20 <20 <20 7 <<100 <20 <20 <20

[0115] d. Pigs. Pigs were immunized with 1 mg env 2-3 with MTP-PE-LO(100 μg MTP-PE) every 21 days. Control animals received the adjuvantalone. Ten days after each immunization the animals were bled, andanti-env 2-3 antibody titers were determined by ELISA. The results inTable 4 show that the two immunized animals developed only low anti-env2 titers (140 and 100, respectively) and no detectable virusneutralizing titers against either the homologous strain (SF2) orheterologous strains (BRU or Zr6). TABLE 4 ELISA and neutralizingantibody titers of swine immunized with env 2-3 MTP-PE-LO adjuvant.^(a)env Neutralizing Immuni- 2-3 titer on: Animal zation ELISA HIV- HIV-HIV- Number Antigen Number titer SF2 BRU Zr6 1371 Env 2-3 pre-^(b)<<50      ^(d)<20     <20 <20 bleed 2 ^(c)<50     <20 <20 <20 3 70<20 <20 <20 4 70 <20 <20 <20 5 80 <20 <20 <20 6 70 <20 <20 <20 7 140 <20<20 <20 1372 Env 2-3 pre- <<50 <20 <20 <20 bleed 2 100 <20 <20 <20 3 70<20 <20 <20 4 70 <20 <20 <20 5 60 <20 <20 <20 6 90 <20 <20 <20 7 90 <20<20 <20 1373 Adjuvant pre- <<50 <20 <20 <20 Control bleed 2 <<50 <20 <20<20 3 <<50 <20 <20 <20 4 <<50 <20 <20 <20 5 <<50 <20 <20 <20 6 <<50 <20<20 <20 7 <<50 <20 <20 <20 1374 Adjuvant pre- <<50 <20 <20 <20 Controlbleed 2 <<50 <20 <20 <20 3 <<50 <20 <20 <20 4 <<50 <20 <20 <20 5 <<50<20 <20 <20 6 <<50 <20 <20 <20 7 <<50 <20 <20 <20

[0116] e. Monkeys. Rhesus macaques were immunized every 30 days with 250μg of env 2-3 with MTP-PE-LO (100 μg MTP-PE). Control animals receivedthe adjuvant formulation alone. One week after each immunization, theanimals were bled and anti-env 2-3 antibody titers were determined byELISA. Table 5 shows that, similar to the dogs, all animals developedantibody titers to env 2-3, but these titers only ranged from 300-3100,far lower than seen previously with guinea pigs. TABLE 5 Titers of env2-3 specific antibodies in sera from Rhesus macaques immunized with env2-3 in MTP-PE-LO adjuvant.^(a) Animal Immunization Antigen NumberPrebleed 1 2 3 4 5 6 Env 2-3 1189 <<100 <<100 300 700 400 400 300 1190<<100 <<100 1,200 800 800 900 500 1191 <<100 <<100 500 2,000 1,300 1,9003,100 1192 <<100 <<100 1,100 900 400 400 500 (average) <<100 <<100 7801,100 700 900 1,100 Adjuvant Control 1197 <<100 <<100 <<100 <<100 <<100<<100 <<100 1198 <<100 <<100 <<100 <<100 <<100 <<100 <<100 1199 <<100<<100 <<100 <<100 <<100 <<100 <<100 1978 <<100 <<100 <<100 <<100 <<100<<100 <<100 (average) <<100 <<100 <<100 <<100 <<100 <<100 <<100

[0117] 2. HSv gD

[0118] a. Goats. A series of adjuvant formulations were tested with gD2in goats. Animals were immunized with 100 μg of gD2 with the variousadjuvants every 21 days. Ten days after the second and thirdimmunizations the animals were bled and anti-gD2 titers were determinedby ELISA. The following adjuvant formulations were used. CFA (1°)followed by IFA (2° & 3°), IFA containing 100 μg MTP-PE), 0.8 mg/mlaluminum hydroxide (alum), MTP-PE-LO (100 μg MTP-PE), MTP-PE-LO-KE (100μg MTP-PE), and MTP-PE-LO-KE (12% squalene, 5.0 mg MTP-PE). The ELISAresults are shown in Table 6. One CFA/IFA animal, both MTP-PE/IFAanimals, and one MTP-PE-LO-KE (5 mg MTP-PE) animal developed highantibody titers (2187-13 172). One CFA/IFA animal, both alum animals,and one MTP-PE-LO-KE (5 mg MTP-PE) animals developed moderate antibodytiters (5691489). The MTP-PE-LO animals and the MTP-PE-LO-KE animalsdeveloped low anti-gD2 titers (46-323). Thus, as with env 2 noted above,the MTP-PE-LO formulation fails to elicit high antibody titers in goats.Modifying the emulsion by using the Kirkland emulsifier (1-2 mm oildroplet sizes) did not improve the adjuvant performance. Vast increasesin MTP-PE (to 5.0 mg) dose appeared to improve the adjuvant performance.TABLE 6 Adjuvant effectiveness with gD2 in the goats. ELISA Titer After2 Immuni- 3 Immuni- Group Animal Adjuvant zations zations 1 3606 CFA/IFA2187 13172 3609 738 770 2 3610 Alum 1489 781 3611 921 522 3 3612MTP-PE-LO 77 194 3613 (100 μg MTP-PE) 145 323 4 3614 MTP-PE-LO-KE 123227 3615 (100 μg MTP-PE) 56 46 5 3624 MTP-PE-LO-KE 142 569 (12%squalene, 615 2291 5.0 mg MTP-PE

[0119] b. Baboons. Juvenile baboons were immunized with gD2 formulatedwith alum, MTP-PE-LO-KE, MTP/IFA and IFA alone. In addition a doseranging study for gD2 combined with alum and MTP-PE-LO-KE was done.Baboons of 2-3 yr (3.4 to 12 kg) were immunized intramuscularly in thethigh three times at three-week intervals. Sera were collected 3 weeksafter the first two immunizations and 2 weeks after the final vaccinedose for determination of gD-specific antibody by ELISA. Whole blood wasdrawn at each of these time points for complete blood cell analyses(CBC). Baboons immunized with 100 μg of gD2 bound to alum developedanti-gD2 mean antibody titers of 3349±550. There was no significantdifference in titers for the three antigen doses tested, 10, 25, 100TABLE 7 HSV vaccine trial in baboons: antibody titers^(a) AdjuvantComposition gD2 ELISA Titers^(b) % of Group (mg) Dose (mg) Dose (mg) 1°Bleed 2° Bleed 3° Bleed MTP-PE/IFA^(c) 1 Alum 400 10 287 (+ 123) 1002 (+366) 1566 (+ 350) 0.6 2 Alum 400 25 1075 (+ 785)   880 (+ 343)  1993 (+1156) 0.8 3 Alum 400 100 720 (+ 184) 1882 (+ 489) 3349 (+ 550) 1.3 4MTP-PE/LO 50 25 140 (+ 63)   788 (+ 331) 1320 (+ 430) 0.5 5 MTP-PE/LO250 10 217 (+ 103) 2490 (+ 995)  3244 (+ 1582) 1.3 6 MTP-PE/LO 250 10057 (+ 34)  925 (+ 254) 2439 (+ 510) 1.0 7 MTP-PE/LO 1000 25 91 (+ 70)1097 (+ 565)  3883 (+ 2401) 1.6 8 MTP-PE/IFA 250 25 24,101 (+ 5423)   62,775 (+ 28,634) 250,382 (+ 64,771) 100 9 IFA 25 2591 (+ 2280)  7631(+ 6563)  66,132 (+ 75,095) 26.4

EXAMPLE 3 MTP-PE-LO Formulation Effective In Stimulating Immunity inLarge Animals

[0120] As demonstrated in Example 2, MTP-PE-LO formulations that wereprepared with a syringe and needle (˜10 micron droplet size) and theKirkland emulsifier (1-2 micron droplet size) failed to give goodimmunostimulation to vaccine antigens in large animals and humans (humandata not shown). The microfluidizer model 110Y was used to generatesmall-droplet-size, stable emulsions. This machine is a high pressure(5000-30,000 PSI) submerged jet type emulsifier. A series of emulsionswere prepared varying in size and stability based on the concentrationsof squalene, Tween 80, and MTP-PE and the physical parameters oftemperature and operating pressure. Examples of different emulsions madewith the microfluidizer are given in Table 8. By changing the physicalparameters and emulsion composition, oil droplet sizes from 1 micron toless than 0.2 microns can be achieved. As demonstrated in Table 8,parameters that decrease emulsion droplet size are increased detergent,increased MTP-PE to squalene ratio, increased operating pressure, andincreased operating temperature. These small droplet size emulsions werethen tested as adjuvants for vaccine antigens in goats and baboons.TABLE 8 Composition and Physical Parameters of MTP-PE-Squalene Emulsionsmade with the Microfluidizer Formu- MTP-PE Squalene Tween 80 MannitolAqueous Temp Pressure Size lation (mg/ml) % % % Phase (° C.) (KPSI) (m)A .01 2 .004 0 H₂O 40° 5 .23 B 0.2 2 .004 0 H₂O 40 5 .17 C 1.0 2 0.16 5H₂O 0 10 .19 D 0.5 2 0 5 H₂O 40 10 .16 E 0.5 2 0 0 H₂O 40 10 .17 F 1.0 40 0 H₂O 30 10 .19 G 1.0 4 0 0 H₂O 20 10 .20 H 1.0 4 0 0 H₂O 0 15 .20 I1.0 4 0 0 H₂O 0 10 .29 J 1.0 4 0 0 H₂O 0 5 .39 K 1.0 4 .16 0 H₂O 0 10.22 L 1.0 4 .016 0 H₂O 0 10 .27 M 1.0 6 0 0 H₂O 0 10 .29

[0121] 1. HSV gD2 in Goats

[0122] The first microfluidizer used with the gD2 antigen was a 4%squalene, 100 μg/ml MTP-PE emulsion without Tween 80 (MTP-PE-LO-MF #13;number designations of MTP-PE-LO-MF formulations are arbitrary and areintended only for use as reference numbers). This material was made atlow pressure in the microfluidizer and had an oil droplet size of about0.8 microns. Goats were immunized intramuscularly with 100 μg of gD2 inthis formulation three times at 21 day intervals. Goats immunized with100 μg gD2, in CFA for primary and IFA for secondary and tertiaryimmunizations served as controls. Ten days after the second and thirdimmunization the animals were bled and anti-gD2 antibody titers weredetermined by ELISA. The results are shown in Table 9. Both animalsreceiving the MTP-PE-LO-MF showed significant anti-gD2 titers. Thesetiters 1661-2966 were intermediate compared to the titers of the twoCFA/IFA control goats (140-24,269). The MTP-PE-LO-MF animals showedtiters that were significantly higher than goats that had receivedMTP-PE-LO formulations prepared in a syringe and needle or in theKirkland emulsifier (see Table 6). In a second experiment in goats, 100μg gD2 was administered every 21 days with MTP-PE-LO-MF #16. Thisformulation consisted of 4% squalene, 500 μg/ml MTP-PE and 0 Tween 80.The oil droplet size of this emulsion was 0.5-0.6 microns. As seen inTable 10, this formulation appeared to give even higher antibody titersthan the previous formulation. Thus, reducing the oil droplet sizeand/or increasing the MTP-PE improves the adjuvant performance of thisemulsion. TABLE 9 Test of MTP-PE-LO-MF #13 as an adjuvant for gD2 inGoats ELISA titer after: Animal 2 Immuni- 3 Immuni- Group NumberAdjuvant Antigen zations zations 1 4519 CFA/IFA gD2 9868 24269 (100 μg)4520 ″ gD2 140 980 (100 μg) 2 4598 MTP-PE- gD2 2966 2207 LO-MF^(a) (100μg) 4599 ″ gD2 1661 N.T.^(b) (100 μg)

[0123] TABLE 10 Test of MTP-PE-LO-MF #13 as an adjuvant for gD2 in GoatsELISA titer after: Animal 2 Immuni- 3 Number Adjuvant Antigen zationszations 5013 MTP-PE-LO-MF gD2 (100 μg) 1299 386 #16 5014 MTP-PE-LO-MFgD2 (100 μg) 6657 2806 #16 5015 MTP-PE-LO-MF gD2 (100 μg) 8206 1943 #165016 MTP-PE-LO-MF gD2 (100 μg) 7886 1514 #16

[0124] 2. HIV env 2-3 and gp120 in Goats.

[0125] Microfluidizer preparations were compared to CFA/IFA and theMTP-PE-LO-KE as adjuvants using the HIV antigen env 2-3 and gp120.Animals were immunized three times at 21-day intervals with 100 μg ofthe gp120 antigen in CFA(1°)/IFA(2° & 3°), MTP-PE-LO-MF #14 (4%squalene, 500 μg/ml MTP-PE, O Tween, phosphate buffered saline)MTP-PE-LO-KE (4% squalene, 100 μg MTP-PE, 0.008% Tween 80, phosphatebuffered saline emulsified in the Kirkland emulsifier) and MTP-PE-LO-MF#15 (4% squalene, 100 μg MTP-PE, 0.008% Tween 80, phosphate bufferedsaline). Animals were also immunized with 100 μg of the HIV antigen env2-3 in CFA/IFA and in MTP-PE-LO-MF #14. The animals were bled 10 daysafter the second and third immunization and anti-env 2-3 antibody titerswere determined by ELISA. The results are shown in Table 11. With env2-3, the animals immunized with the MTP-PE-LO-MF #14 formulation showedequivalent titer to CFA/IFA animals after two immunizations and highertiters than the CFA/IFA animals after three immunizations. With gp120the results were not quite as clear. The MTP-PE-LO-MF #14 animals showmuch more variation than the CFA/IFA animals. Thus the mean titers forthe microfluidizer group is lower than the CFA group, but individualanimals receiving MTP-PE-LO-MF #14 did show titers as high as anyanimals in the CFA/IFA group. A direct comparison with gp120 ofidentical adjuvant components (4% squalene, 100 μg/ml MTP-PE, 0.008%Tween 80, phosphate buffered saline) emulsified by two different methods(Kirkland emulsifier vs. microfluidizer) illustrates the importance ofthe small droplet size in the emulsion. The Kirkland emulsifier groupshowed mean titer of 632 after these immunizations while themicrofluidizer group showed mean titer of 3277. TABLE 11 Test ofMTP-PE-LO-MF as an adjuvant with HIV antigens env 2 and gp120 ELISATiter after: Animal 2 immuni- Genometric 3 immuni- Genometric GroupNumber Adjuvant Antigen zation Mean + SE zation Mean + SE 1 5018 CFA/IFAgp120 (100 mg) 900 1861 + 539 7300 6630 + 996 5019 ″ gp120 (100 mg) 37005700 5020 ″ gp120 (100 mg) 2000 7100 5021 ″ gp120 (100 mg) 1800 3400 25022 CFA/IFA env 2 (100 mg) 2400 3000 5023 ″ env 2 (100 mg) 4600 2235 +680 3400  5074 + 1378 5024 ″ env 2 (100 mg) 2400 8900 3 5026MTP-PE-LO-MF gp120 (100 mg) 0 800 #14^(a) 5027 ″ gp120 (100 mg) 300 101 + 1089 500 1324 + 994 5029 ″ gp120 (100 mg) 3407 5800 4 5030MTP-PE-LO-MF env 2 (100 mg) 7900 19,500 #14^(a) 5031 ″ env 2 (100 mg)4600  2351 + 1688 6600  9896 + 2493 5032 ″ env 2 (100 mg) 300 6900 5033″ env 2 (100 mg) 2800 10,800 5 5034 MTP-PE-LO-KE^(b) gp120 (100 mg) 0600 5035 ″ gp120 (100 mg) 1400  721 + 416 600 632 + 32 5037 ″ gp120 (100mg) 400 700 6 5038 MTP-PE-LO-MF gp120 (100 mg) 1000 5100 #15^(c) 5040 ″gp120 (100 mg) 0  10 + 333 2300 3277 + 767 5041 ″ gp120 (100 mg) 0 3000

[0126] 3. HIV env 2-3 and gp120 in baboons.

[0127] MTP-PE-LO-MF #1 (2% squalene, 500 μg/ml MTP-PE, O Tween 80, H20,oil droplet size ˜0.17 microns) was tested as an adjuvant with the HIVantigens env 2-3 and gp120 in baboons. MTP-PE in IFA and alum were usedas controls. Animals were immunized at one month intervals. Two weeksafter the second immunization, the animals were bled and anti-env 2-3antibody virus neutralizing titers were determined. The results areshown in Table 12. Antibody titers against gp120 were higher withMTP-PE-LO-MF #1 than with MTP-PE-IFA. Anti-env 2-3 titers were similarin the MTP-PE-IFA and MTP-PE-LO-MF #1 groups. Anti-gp120 titers achievedwith alum were in the same range as with MTP-PE-LO-MF. #1 but anti env2-3 titers achieved with alum appear lower than with the MTP-PEadjuvants. TABLE 12 Test of MTP-PE-LO-MF #1 as an Adjuvant for HIVProtein env2 and gp120 in Baboons Virus ELISA Animal Titer After 2Neutralizing Group Number Adjuvant Antigen Immunizations Antibody Titer1 2947 MTP/IFA gp120 (55 mg) <100 <10 2948 (350 mgMTP-PE) gp120 (55 mg)<100 <10 2949 ″ gp120 (55 mg) 3000 <10 2 2550 MTP-PE/IFA   env2 (25 mg)400 <10 2451 (250 mgMTP-PE)   env2 (25 mg) 34,500 30 2952 ″   env2 (25mg) 142,300 200 3 2953 MTP-PE-LO-MF #1^(a) gp120 (55 mg) 51,000 200 2957″ gp120 (55 mg) 43,000 35 2595 ″ gp120 (55 mg) 800 50 4 2956MTP-PE-LO-MF #1   env2 (25 mg) 600 <10 2957 ″   env2 (25 mg) 14,400 352958 ″   env2 (25 mg) 87,400 >250 5 2964 Alum^(b) gp120 (55 mg) 56,000150 2965 ″ gp120 (55 mg) 100 <10 6 2966 Alum   env2 (25 mg) 4900 80 ″  env2 (25 mg) 700 <10

EXAMPLE 4 Additional Adjuvant/Antigen Formulations

[0128] In addition to the detailed examples set forth above, a number ofother antigens have been prepared in vaccine formulations containingadjuvant compositions of the invention. These include antigens frompathogens responsible for influenza and malaria, as well as antigensassociated with HIV and HSV other than those described in previousexamples. Antigens from cytomegalovirus (CMV) and hepatitis C virus(HCV) are also described, as these antigens can be used in the sameadjuvant formulations described for the other indicated antigens.

[0129] Antigens

[0130] Influenza antigens suitable for use in vaccine preparations arecommercially available. Antigens used in the following examples areFluogen®, manufactured by Parke-Davis; Duphar, manufactured by Duphar B.V.; and influenza vaccine batch A41, manufactured by InstitutoVaccinogeno Pozzi.

[0131] Malaria antigens suitable for use in vaccine preparations aredescribed in U.S. patent application Ser. No. 336,288, filed Apr. 11,1989, and in U.S. Pat. No. 4,826,957, issued May 2, 1989.

[0132] Additional HIV antigens suitable for use in vaccine preparationsare described in U.S. application Ser. No. 490,858, filed Mar. 9, 1990.Also see published European application number 181150 (May14, 1986) foradditional HIV antigens.

[0133] Additional HSV antigens suitable for use in vaccine preparationsare described in PCT WO85/04587, published Oct. 24, 1985, and PCTWO88/02634, published Apr. 21, 1988. Mixtures of gB and gD antigens,which are truncated surface antigens lacking the anchor regions, areparticularly preferred.

[0134] Cytomegalovirus antigens suitable for use in vaccine preparationsare described in U.S. Pat. No. 4,689,225, issued Aug. 25, 1987, and inPCT application PCT/US89/00323, published Aug. 10, 1989 underInternational Publication Number WO 89/07143. Also see U.S. applicationSer. NO. 367,363, filed Jun. 16, 1989.

[0135] Hepatitis C antigens suitable for use in vaccine preparations aredescribed in PCT/US88/04125, published European application number318216 (May 31, 1989), published Japanese application number 1-500565(filed Nov. 18, 1988), and Canadian application 583,561. A different setof HCV antigens is described in European patent application 90/302866.0,filed Mar. 16, 1990. Also see U.S. application Ser. No. 456,637, filedDec. 21, 1989, and PCT/US90/01348.

[0136] It should be noted that published versions of the variousunpublished application numbers listed above can be obtained from anindexing service such as World Patent Index, as well as a listing ofcorresponding applications in other countries.

[0137] Adjuvant Formulations and Preparation Techniques

[0138] The following summaries describe adjuvant formulations and howthey are prepared as well as vaccine compositions prepared using theadjuvants and various antigenic substances. In some cases summaries ofvaccination studies are provided, but without the detail of the examplesabove, since the vaccination studies set forth above already providesufficient guidance for use of the vaccine compositions.

[0139] Influenza

[0140] In a series of experiments, hamsters were immunized with acommercial influenza vaccine from Instituto Vaccinogeno Pozzi. Thisvaccine consists of purified HA from two A strains (A/Leningrad/360/86and A/Singapore/6/86) and one B strain (B/Ann Arbor/1/86). The vaccinewas tested alone, with an MTP-PE/LO emulsion made with a Kirklandemulsifier (Fluoromed Pharmaceutical, Inc., La Mesa, Calif.) and with anMTP-PE/MF emulsion made in a microfluidizer (model 110Y, Microfluidics,Newton, Mass.). The first two are comparative compositions, while the“MF” composition is a composition of the invention. MTP-PE/MF stands for“MTP-PE Microfluidizer” emulsion and contains 4% squalene and 1.0 mg/mlMTP-PE emulsified with the Microfluidizer. The MTP-PE Kirkland emulsioncontained 4% squalene, 0.5 mg/ml MTP-PE, and 0.008% Tween 80 emulsifiedwith the Kirkland emulsifier. Animals received three immunizationscontaining 8.3 μg of each HA antigen. MTP-PE was used at 50 μg per dosein both formulations. ELISA titers were determined against theimmunizing antigens after each immunization and HAI titers weredetermined after the second immunization. ELISA titers were increasedsubstantially by both of the adjuvant formulations tested.

[0141] In other experiments, hamsters were immunized with either thecommercially available Parke-Davis Fluogen vaccine (HA A/Shanghai/11/87,A/Taiwan/1/86 and B/Yamagata/16/88) or the commercially available Dupharinfluenza vaccine (HA A/Sechuan/2/87, A/Singapore/6/86 andB/Beijing/1/87) alone or with the MF69 adjuvant formulation (MF69 is 5%squalene, 0.2% Tween 80, 0.8%, Span 85, and 400 μg/ml MTP-PE, emulsifiedin the Microfluidizer). Equal volumes of vaccine were mixed with theMF69 adjuvant. Animals received three immunizations of 11.25 μg of theParke-Davis vaccine or 7.5 μg of the Duphar vaccine at three weekintervals. Animals receiving the MF69 adjuvant received 50 μg doses ofMTP-PE. The animals receiving Duphar plus MF69 showed significantlyhigher anti-HA titers than Duphar alone after one and two immunizations(mean titers 80-fold higher than vaccine alone after one immunizationand 170-fold higher than after two immunizations). The MF69 adjuvantshowed good stimulation of antibody response to the Parke-Davis vaccine,generating mean titers of 2951, 14,927 and 12,878 after one, two orthree immunizations. This represents titers 82, 29 and 10-fold higherthan vaccine alone after one, two or three immunizations, respectively.For both vaccines, peak antibody titers were seen after twoimmunizations with MF 69.

[0142] In further experiments, the immunogenicity of two commercialinfluenza vaccines, Parke-Davis Fluogen and Duphar subunit influenza,were compared with no adjuvant and with several MTP-PE containingadjuvant formulations in goats. The animals were immunizedintramuscularly with 0.5 ml of each vaccine mixed with either 0.5 ml ofPBS or 0.5 ml of MTP-PE adjuvant formulations. Three adjuvantformulations were compared: 200 μg of MTP-PE dissolved in PBS, and 200μg of MTP-PE in two different microfluidized emulsions, referred to asGaulin ¼ and MF40/4 emulsions. Gaulin ¼ consists of 1.6% squalene and400 μg/ml MTP-PE emulsified in the Gaulin homogenizer (APV Gaulin,Everett, Mass.). MTP-PE/MF-40/4 consists of 1.6% squalene, 400 μg/mlMTP-PE, 0.154% Tween 85, and 0.166% Span 85 emulsified in theMicrofluidizer (Model 110Y, Microfluidics, Newton, Mass.). Animalsreceived 0.5 ml of vaccine mixed with either 0.5 ml of PBS or 0.5 ml ofthe indicated adjuvant formulation to generate a 1.0 ml injectionvolume. As with the hamsters, the goats receiving the influenza vaccinescombined with the adjuvant emulsions showed much higher antibody titersthan goats receiving vaccine alone. This is especially pronounced earlyin the immunization schedule. After one immunization the Gaulin ¼emulsion generated anti-HA titers greater than 30-fold higher than theParke-Davis vaccine alone. The MTP-PE/MF-40 emulsion generated anti-HAtiters that were greater than 130-fold higher than Parke-Davis vaccinealone and 60-fold higher than Duphar vaccine alone. MTP-PE in PBS showedno stimulation of antibody titer after one immunization. After twoimmunizations, similar increases in antibody titers with the emulsionswere seen. The early stimulation of anti-HA titers seen with theadjuvant emulsions is especially significant since influenza vaccinesare generally given as one dose vaccines to adults and two dose vaccinesto infants. Thus, as in hamsters, the MTP-PE-emulsions show largeincreases in the immune response to influenza vaccines.

[0143] In another experiment, the Duphar vaccine was compared alone andwith adjuvant formulation MF69. The Parke-Davis vaccine was comparedalone and with MF101, MF69, MF-68+MTP-PE, and the Ribi Adjuvant systemmade in the Gaulen homogenizer (micro-fluidizer). MF-101 consists of1.6% squalene and 400 ug/ml MTP-PE, emulsified in the Microfluidizer.MF-68 consists of 5% squalene, 0.8% Span 85, and 0.2% Tween 80,emulsified in the Microfluidizer. MF-68+MTP consists of MF-68 to whichwas added 400 ug/ml MTP-PE per ml post emulsification. Ribi-MF consistsof 2% squalene, 0.4% Tween 20, 250 ug/ml monophosphoryl lipid A, 250ug/ml Trehalose dimycolate, and 250 ug/ml cell wall skeleton (RibiImmunochem, Hamilton Mont.), emulsified in the Gaulin homogenizer. Alladjuvants were used at a dose of 0.5 ml per injection with equal volumesof vaccine (antigen). MF69 significantly increased the ELISA titer tothe Duphar vaccine. All of the adjuvants tested also significantlyincreased the immunogenicity of the Parke-Davis vaccine as measured byboth ELISA titer and hemagglutination titer.

[0144] In a further experiment, MF69 and MF59 formulations (differingonly in the Tween 80:Span 85 ratio; see descriptions above) werecompared as adjuvants with the Parke-Davis influenza vaccine in goats.The animals were immunized once with one-half of the human vaccine dose(7.5 μg each of the three HA components) combined with the adjuvantformulations. MTP-PE was used at a dose of 100 μg in the formulations.As expected, the two formulations give very similar titers with the MF69showing a mean titer of 926 and the MF59 showing a mean titer of 821.

[0145] Malaria

[0146] A vaccination study has been initiated using MF59 (describedabove) as adjuvant. A mixture of commercially available antigens fromthe sporozoite, merozoite, and erythrocytic stages of the disease wasused: Falc. 2.3 circumsporozoite antigen, HP 195 merozoite antigen, andSERA 1 red blood stage antigen. Vaccine compositions are prepared asdescribed above, namely mixing equal volumes of the previously preparedMF59 adjuvant and the antigen composition.

[0147] HIV

[0148] An immunization experiment was carried out to compare productionof neutralizing antibodies by a number of different gp120 antigens.Details of preparation of the antigens are set forth in U.S. applicationSer. No. 490,858, filed Mar. 9, 1990. One antigen was a gp120 analog(env 2-3) prepared in yeast, which is denatured and non-glycosylated.Another antigen was glycosylated gp120 retaining its naturalconfiguration. Both gp120 materials were derived from the same genesource, HIV-1 SF-2 isolate. Antibody production was measured in baboons.Initial studies using oil-containing adjuvants with particle sizeslarger than 1 micron produced titers less than those produced usingconventional alum adjuvants. However, later studies with submicronparticle adjuvants produced antibody titers at least 10-fold higher thanwith alum. The initial submicron composition contained 2% squalene and0.500 mg/ml MTP-PE in water and had oil droplets averaging about 0.17microns in diameter. Vaccine compositions using MF59 (described above)or MF58 (MF59 but with MTP-PE added exogenously) as an adjuvant inbaboons have proven even more effective in stimulating antibodyproduction than the initial submicron composition used. MF59 was used ata 1:2 dilution at a rate of 0.100 mg MTP-PE.

[0149] Herpes Simplex Virus

[0150] In addition to the gD2 experiments described above, additionalexperiments have been carried out using MF59 and various amounts ofMTP-PE and antigens. Satisfactory antibody tiers have been obtainedusing from 0.003 to 0.250 mg gD2 with MF59 adjuvant and 0.050 mg MTP-PEin guinea pigs (intramuscular administration) and using from 0.010 to0.100 mg gD2 with MF59 and 0.100 mg MTP-PE.

[0151] Cytomegalovirus

[0152] Vaccine formulations can be prepared by mixing from 0.001 to0.250 mg of CMV antigens in 0.5 ml physiological saline with 0.5 ml MF59adjuvant containing 0.050 mg MTP-PE. MF69, MF101, and other submicronparticle adjuvants can be used in the same manner.

[0153] Hepatitis C Virus

[0154] Vaccine formulations can be prepared by mixing from 0.001 to0.250 mg of HCV antigens in 0.5 ml physiological saline with 0.5 ml MF59adjuvant containing 0.050 mg MTP-PE. MF69, MF101, and other submicronparticle adjuvants can be used in the same manner.

[0155] All publications and patent applications cited herein areincorporated by reference in the location where cited to the same extentas if each individual publication or patent application had beenindividually indicated to be incorporated by reference.

[0156] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. An adjuvant composition, comprising: (1) ametabolizable oil and (2) an emulsifying agent, wherein said oil andsaid emulsifying agent are present in the form of an oil-in-wateremulsion having oil droplets substantially all of which are less than 1micron in diameter and wherein said composition exists in the absence ofany polyoxypropylene-polyoxyethylene block copolymer.
 2. The compositionof claim 1, wherein said oil is an animal oil.
 3. The composition ofclaim 2, wherein said oil is an unsaturated hydrocarbon.
 4. Thecomposition of claim 1, wherein said oil is a terpenoid.
 5. Thecomposition of claim 1, wherein said oil is a vegetable oil.
 6. Thecomposition of claim 1, wherein said composition comprises 0.5 to 20% byvolume of said oil in an aqueous medium.
 7. The composition of claim 1,wherein said emulsifying agent comprises a non-ionic detergent.
 8. Thecomposition of claim 20, wherein said emulsifying agent comprises apolyoxyethylene sorbitan mono-, di-, or triester or a sorbitan mono-,di-, or triether.
 9. The composition of claim 8, wherein saidcomposition comprises 0.01 to 0.5% by weight of said emulsifying agent.10. The composition of claim 9, wherein said composition furthercomprises a separate immunostimulating agent.
 11. The composition ofclaim 8, wherein said immunostimulating agent comprises alum or abacterial cell wall component.
 12. The composition of claim 11, whereinsaid composition comprises 0.0001 to 1.0% by weight of saidimmunostimulating agent.
 13. The composition of claim 11, wherein saidimmunostimulating agent comprises a muramyl peptide.
 14. The compositionof claim 1, wherein said emulsifying agent also functions as animmunostimulating agent.
 15. The composition of claim 14, wherein saidcomposition comprises 0.01 to 0.5% by weight of said immunostimulatingagent.
 16. The composition of claim 14, wherein said immunostimulatingagent comprises a lipophilic muramyl peptide.
 17. The composition ofclaim 16, wherein said peptide comprises a muramyl dipeptide or amuramyl tripeptide.
 18. The composition of claim 17, wherein saidpeptide further comprises a phospholipid.
 19. The composition of claim18, wherein said phospholipid comprises a phosphoglyceride.
 20. Thecomposition of claim 14, wherein said peptide is a compound of theformula

wherein R is H or COCH₃; R¹, R², and R³ independently represent H or alipid moiety; R⁴ is hydrogen or alkyl; X and Z independently representan aminoacyl moiety selected from the group consisting of alanyl, valyl,leucyl, isoleucyl, α-aminobutyryl, threonyl, methionyl, cysteinyl,glutamyl, isoglutamyl, glutaminyl, isoglutaminyl, aspartyl,phenylalanyl, tyrosyl, tryptophanyl, lysyl, ornithinyl, arginyl,histidyl, asparaginyl, prolyl, hydroxypropyl, seryl, and glycyl; n is 0or 1; Y is —NHCHR⁵CH₂CH₂CO—, wherein R⁵ represents an optionallyesterified or amidated carboxyl group; and L is OH, NR⁶R⁷ where R⁶ andR⁷ independently represent H or a lower alkyl group, or a lipid moiety.21. The composition of claim 20, wherein R⁴ is methyl, X is alanyl, andY is isoglutaminyl.
 22. The composition of claim 20, wherein n is 1; Zis alanyl; R is acetyl; and R¹, R², and R³ are all H.
 23. Thecomposition of claim 22, wherein L comprises a phospholipid moiety. 24.The composition of claim 23, wherein said phospholipid moiety comprisesa diacylphosphoglyceride.
 25. The composition of claim 20, wherein saidpeptide isN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1,2-dipalmitoyl-sn-glycero-3-(hydroxy-phosphoryloxy)]ethylamide.26. The composition of claim 20, wherein at least one of R¹ and R²represents an acyl group containing from 1 to 22 carbons.
 27. Thecomposition of claim 20, wherein at least one of R¹, R², and R³represents an acyl group containing from 14 to 22 carbons.
 28. A vaccinecomposition, comprising: (1) an immunostimulating amount of an antigenicsubstance, and (2) an immunostimulating amount of the adjuvant ofclaim
 1. 29. A method of stimulating an immune response in a hostanimal, comprising: administering a protective antigen to said animal inthe presence of an immunostimulating amount of submicron metabolizableoil droplets in a continuous aqueous phase and in the absence of anypolyoxypropylene-polyoxyethylene block copolymer.
 30. The method ofclaim 29, wherein said oil droplets further comprise an emulsifyingagent.
 31. The method of claim 30, wherein said oil droplets furthercomprise an immunostimulating agent separate from said oil and saidemulsifying agent.
 32. The method of claim 31, wherein saidimmuno-stimulating agent comprises alum or a bacterial cell wallcomponent.
 33. The method of claim 31, wherein said immuno-stimulatingagent comprises a muramyl peptide.
 34. The method of claim 30, whereinsaid emulsifying agent is also effective as an immunostimulating agent.35. The method of claim 34, wherein said immuno-stimulating agentcomprises a lipophilic muramyl peptide.